title: Caspase activation as a versatile assay platform for detection of cytotoxic bacterial toxins.
abstract: Pathogenic bacteria produce several virulence factors that help them establish infection in permissive hosts. Bacterial toxins are a major class of virulence factors and hence are attractive therapeutic targets for vaccine development. Here, we describe the development of a rapid, sensitive, and high-throughput assay that can be used as a versatile platform to measure the activities of bacterial toxins. We have exploited the ability of these toxins to cause cell death via apoptosis of sensitive cultured cell lines as a readout for measuring toxin activity. Caspases (cysteine-aspartic proteases) are induced early in the apoptotic pathway, and so we used their induction to measure the activities of Clostridium difficile toxins A (TcdA) and B (TcdB) and binary toxin (CDTa-CDTb), Corynebacterium diphtheriae toxin (DT), and Pseudomonas aeruginosa exotoxin A (PEA). Caspase induction in the cell lines, upon exposure to toxins, was optimized by toxin concentration and intoxication time, and the specificity of caspase activity was established using a genetically mutated toxin and a pan-caspase inhibitor. In addition, we demonstrate the utility of the caspase assay for measuring toxin potency, as well as neutralizing antibody (NAb) activity against C. difficile toxins. Furthermore, the caspase assay showed excellent correlation with the filamentous actin (F-actin) polymerization assay for measuring TcdA and TcdB neutralization titers upon vaccination of hamsters. These results demonstrate that the detection of caspase induction due to toxin exposure using a chemiluminescence readout can support potency and clinical immunogenicity testing for bacterial toxin vaccine candidates in development.
authors.affiliation: Vaccines Basic Research, Merck Research Laboratories, West Point, Pennsylvania, USA. angela_payne2@merck.com
authors.forename: Angela M
authors.initials: AM
authors.lastname: Payne
authors.forename: Julie
authors.initials: J
authors.lastname: Zorman
authors.forename: Melanie
authors.initials: M
authors.lastname: Horton
authors.forename: Sheri
authors.initials: S
authors.lastname: Dubey
authors.forename: Jan
authors.initials: J
authors.lastname: ter Meulen
authors.forename: Kalpit A
authors.initials: KA
authors.lastname: Vora
mesh_terms: D000818:Animals; D017209:Apoptosis; D001427:Bacterial Toxins / Q000032:analysis* / Q000633:toxicity; D020169:Caspases / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003584:Cytological Techniques / Q000379:methods*; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods; D014709:Vero Cells
chemical_list: D001427:Bacterial Toxins; D020169:Caspases